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Abstract: Background and Aim: rdxA and frxA mutations and enhancement of efflux pump have been suggested as the cause of metronidazole resistance in Helicobacter pylori. This study was performed to investigate the resistance mechanisms related to clinical eradication outcome, and to examine direct involvement of hefA in metronidazole‐resistant isolates with intact rdxA and frxA. Methods: A total of 53 H. pylori‐positive patients who were treated with metronidazole‐containing sequential or quadruple therapy from 2011 to 2015 were enrolled. The metronidazole susceptibility of H. pylori isolates was examined by agar dilution test. Mutations in rdxA and frxA, were analyzed with DNA sequencing, and impact of hefA on metronidazole resistance was examined with quantitative real‐time reverse transcription polymerase chain reaction, knockout and genetic complementation test for hefA. Results: Seven mutation types of rdxA and/or frxA were found in H. pylori isolated from non‐eradicated subjects. rdxA mutation was associated with eradication failure (P = 0.002), and nonsense mutation in rdxA reduced eradication efficacy (P = 0.009). hefA expression was significantly higher in resistant isolates (P < 0.001), especially in rdxA(−)frxA(−) as compared to rdxA(+)frxA(+) (P = 0.027). Resistant isolates with no mutation in rdxA and frxA became susceptible after hefA knockout. Genetic complementation for hefA recovered metronidazole resistance in all of three hefA knockout mutants. Conclusions: These results suggest that rdxA mutations play a critical role in metronidazole resistance as well as the outcomes of eradication therapy. In addition, hefA seems to be directly involved in metronidazole resistance, which explains the resistance in clinical isolates with intact rdxA and frxA. [ABSTRACT FROM AUTHOR]